Neurodegeneration and Epilepsy in a Zebrafish Model of CLN3 Disease (Batten Disease)

The neuronal ceroid lipofuscinoses are a group of lysosomal storage disorders that comprise the most common, genetically heterogeneous, fatal neurodegenerative disorders of children. They are characterised by childhood onset, visual failure, epileptic seizures, psychomotor retardation and dementia. CLN3 disease, also known as Batten disease, is caused by autosomal recessive mutations in the CLN3 gene, 80–85% of which are a ~1 kb deletion. Currently no treatments exist, and after much suffering, the disease inevitably results in premature death. The aim of this study was to generate a zebrafish model of CLN3 disease using antisense morpholino injection, and characterise the pathological and functional consequences of Cln3 deficiency, thereby providing a tool for future drug discovery. The model was shown to faithfully recapitulate the pathological signs of CLN3 disease, including reduced survival, neuronal loss, retinopathy, axonopathy, loss of motor function, lysosomal storage of subunit c of mitochondrial ATP synthase, and epileptic seizures, albeit with an earlier onset and faster progression than the human disease. Our study provides proof of principle that the advantages of the zebrafish over other model systems can be utilised to further our understanding of the pathogenesis of CLN3 disease and accelerate drug discovery

Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome

Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome wide siRNA screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies

Comparative Influence of Ocean Conditions on Yellowfin and Atlantic Bluefin Tuna Catch from Longlines in the Gulf of Mexico

Directed fishing effort for Atlantic bluefin tuna in the Gulf of Mexico (GOM), their primary spawning grounds in the western Atlantic, has been prohibited since the 1980s due to a precipitous decline of the spawning stock biomass. However, pelagic longlines targeted at other species, primarily yellowfin tuna and swordfish, continue to catch Atlantic bluefin tuna in the GOM as bycatch. Spatial and temporal management measures minimizing bluefin tuna bycatch in the GOM will likely become important in rebuilding the western Atlantic bluefin stock. In order to help inform management policy and understand the relative distribution of target and bycatch species in the GOM, we compared the spatiotemporal variability and environmental influences on the catch per unit effort (CPUE) of yellowfin (target) and bluefin tuna (bycatch). Catch and effort data from pelagic longline fisheries observers (1993–2005) and scientific tagging cruises (1998–2002) were coupled with environmental and biological data. Negative binomial models were used to fit the data for both species and Akaike's Information Criterion (corrected for small sample size) was used to determine the best model. Our results indicate that bluefin CPUE had higher spatiotemporal variability as compared to yellowfin CPUE. Bluefin CPUE increased substantially during the breeding months (March-June) and peaked in April and May, while yellowfin CPUE remained relatively high throughout the year. In addition, bluefin CPUE was significantly higher in areas with negative sea surface height anomalies and cooler sea surface temperatures, which are characteristic of mesoscale cyclonic eddies. In contrast, yellowfin CPUE was less sensitive to environmental variability. These differences in seasonal variability and sensitivity to environmental influences suggest that bluefin tuna bycatch in the GOM can be reduced substantially by managing the spatial and temporal distribution of the pelagic longline effort without substantially impacting yellowfin tuna catches

A Genome-Wide Association Study of Diabetic Kidney Disease in Subjects With Type 2 Diabetes

dentification of sequence variants robustly associated with predisposition to diabetic kidney disease (DKD) has the potential to provide insights into the pathophysiological mechanisms responsible. We conducted a genome-wide association study (GWAS) of DKD in type 2 diabetes (T2D) using eight complementary dichotomous and quantitative DKD phenotypes: the principal dichotomous analysis involved 5,717 T2D subjects, 3,345 with DKD. Promising association signals were evaluated in up to 26,827 subjects with T2D (12,710 with DKD). A combined T1D+T2D GWAS was performed using complementary data available for subjects with T1D, which, with replication samples, involved up to 40,340 subjects with diabetes (18,582 with DKD). Analysis of specific DKD phenotypes identified a novel signal near GABRR1 (rs9942471, P = 4.5 x 10(-8)) associated with microalbuminuria in European T2D case subjects. However, no replication of this signal was observed in Asian subjects with T2D or in the equivalent T1D analysis. There was only limited support, in this substantially enlarged analysis, for association at previously reported DKD signals, except for those at UMOD and PRKAG2, both associated with estimated glomerular filtration rate. We conclude that, despite challenges in addressing phenotypic heterogeneity, access to increased sample sizes will continue to provide more robust inference regarding risk variant discovery for DKD.Peer reviewe

Identification of TRPC6 as a possible candidate target gene within an amplicon at 11q21-q22.2 for migratory capacity in head and neck squamous cell carcinomas

Abstract: Background: Cytogenetic and gene expression analyses in head and neck squamous cell carcinomas (HNSCC) have allowed identification of genomic aberrations that may contribute to cancer pathophysiology. Nevertheless, the molecular consequences of numerous genetic alterations still remain unclear. Methods: To identify novel genes implicated in HNSCC pathogenesis, we analyzed the genomic alterations present in five HNSCC-derived cell lines by array CGH, and compared high level focal gene amplifications with gene expression levels to identify genes whose expression is directly impacted by these genetic events. Next, we knocked down TRPC6, one of the most highly amplified and over-expressed genes, to characterize the biological roles of TRPC6 in carcinogenesis. Finally, real time PCR was performed to determine TRPC6 gene dosage and mRNA levels in normal mucosa and human HNSCC tissues. Results: The data showed that the HNSCC-derived cell lines carry most of the recurrent genomic abnormalities previously described in primary tumors. High-level genomic amplifications were found at four chromosomal sites (11q21-q22.2, 18p11.31-p11.21, 19p13.2-p13.13, and 21q11) with associated gene expression changes in selective candidate genes suggesting that they may play an important role in the malignant behavior of HNSCC. One of the most dramatic alterations of gene transcription involved the TRPC6 gene (located at 11q21-q22.2) which has been recently implicated in tumour invasiveness. siRNA-induced knockdown of TRPC6 expression in HNSCC-derived cells dramatically inhibited HNSCC-cell invasion but did not significantly alter cell proliferation. Importantly, amplification and concomitant overexpression of TRPC6 was also found in HNSCC tumour samples. Conclusions: Altogether, these data show that TRPC6 is likely to be a target for 11q21-22.2 amplification that confers enhanced invasive behavior to HNSCC cells. Therefore, TRPC6 may be a promising therapeutic target in the treatment of HNSCC.This work was supported by Instituto de Salud Carlos III-Fondo de Investigacion Sanitaria [FIS PI11/929 to M.-D.C and C. S.]; Red Tematica de Investigacion Cooperativa en Cancer [RD12/0036/0015] Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness & European Regional Development Fund (ERDF); and Obra Social CajAstur-Instituto Universitario de Oncologia del Principado de Asturias.Bernaldo De Quirós, S.; Merlo, A.; Secades, P.; Zambrano, I.; Saenz De Santa María, I.; Ugidos, N.; Jantus Lewintre, E.... (2013). Identification of TRPC6 as a possible candidate target gene within an amplicon at 11q21-q22.2 for migratory capacity in head and neck squamous cell carcinomas. BMC Cancer. 13(116):1-9. https://doi.org/10.1186/1471-2407-13-116S1913116Akervall J: Genomic screening of head and neck cancer and its implications for therapy planning. Eur Arch Otorhinolaryngol. 2006, 263: 297-304. 10.1007/s00405-006-1039-1.Squire JA, Bayani J, Luk C, Unwin L, Tokunaga J, MacMillan C, Irish J, Brown D, Gullane P, Kamel-Reid S: Molecular cytogenetic analysis of head and neck squamous cell carcinoma: by comparative genomic hybridization, spectral karyotyping, and expression array analysis. Head Neck. 2002, 24: 874-887. 10.1002/hed.10122.Perez-Ordonez B, Beauchemin M, Jordan RC: Molecular biology of squamous cell carcinoma of the head and neck. J Clin Pathol. 2006, 59: 445-453. 10.1136/jcp.2003.007641.Tan KD, Zhu Y, Tan HK, Rajasegaran V, Aggarwal A, Wu J, Wu HY, Hwang J, Lim DT, Soo KC, Tan P: Amplification and overexpression of PPFIA1, a putative 11q13 invasion suppressor gene, in head and neck squamous cell carcinoma. Genes Chromosomes Cancer. 2008, 47: 353-362. 10.1002/gcc.20539.Rodrigo JP, Garcia LA, Ramos S, Lazo PS, Suarez C: EMS1 Gene amplification correlates with poor prognosis in squamous cell carcinomas of the head and neck. Clin Cancer Res. 2000, 6: 3177-3182.Callender T, el-Naggar AK, Lee MS, Frankenthaler R, Luna MA, Batsakis JG: PRAD-1 (CCND1)/cyclin D1 oncogene amplification in primary head and neck squamous cell carcinoma. Cancer. 1994, 74: 152-158. 10.1002/1097-0142(19940701)74:13.0.CO;2-K.Huang X, Gollin SM, Raja S, Godfrey TE: High-resolution mapping of the 11q13 amplicon and identification of a gene, TAOS1, that is amplified and overexpressed in oral cancer cells. Proc Natl Acad Sci U S A. 2002, 99: 11369-11374. 10.1073/pnas.172285799.Gorogh T, Weise JB, Holtmeier C, Rudolph P, Hedderich J, Gottschlich S, Hoffmann M, Ambrosch P, Csiszar K: Selective upregulation and amplification of the lysyl oxidase like-4 (LOXL4) gene in head and neck squamous cell carcinoma. J Pathol. 2007, 212: 74-82. 10.1002/path.2137.Begum A, Imoto I, Kozaki K, Tsuda H, Suzuki E, Amagasa T, Inazawa J: Identification of PAK4 as a putative target gene for amplification within 19q13.12-q13.2 In oral squamous-cell carcinoma. Cancer Sci. 2009, 100: 1908-1916. 10.1111/j.1349-7006.2009.01252.x.Secades P, Rodrigo JP, Hermsen M, Alvarez C, Suarez C, Chiara MD: Increase in gene dosage is a mechanism of HIF-1alpha constitutive expression in head and neck squamous cell carcinomas. Genes Chromosomes Cancer. 2009, 48: 441-454. 10.1002/gcc.20652.Singh B, Gogineni SK, Sacks PG, Shaha AR, Shah JP, Stoffel A, Rao PH: Molecular cytogenetic characterization of head and neck squamous cell carcinoma and refinement of 3q amplification. Cancer Res. 2001, 61: 4506-4513.Baldwin C, Garnis C, Zhang L, Rosin MP, Lam WL: Multiple microalterations detected at high frequency in oral cancer. Cancer Res. 2005, 65: 7561-7567.Roman E, Meza-Zepeda LA, Kresse SH, Myklebost O, Vasstrand EN, Ibrahim SO: Chromosomal aberrations in head and neck squamous cell carcinomas in Norwegian and Sudanese populations by array comparative genomic hybridization. Oncol Rep. 2008, 20: 825-843.Weber RG, Sommer C, Albert FK, Kiessling M, Cremer T: Clinically distinct subgroups of glioblastoma multiforme studied by comparative genomic hybridization. Lab Invest. 1996, 74: 108-119.Knuutila S, Bjorkqvist AM, Autio K, Tarkkanen M, Wolf M, Monni O, Szymanska J, Larramendy ML, Tapper J, Pere H: DNA copy number amplifications in human neoplasms: review of comparative genomic hybridization studies. Am J Pathol. 1998, 152: 1107-1123.Menghi-Sartorio S, Mandahl N, Mertens F, Picci P, Knuutila S: DNA copy number amplifications in sarcomas with homogeneously staining regions and double minutes. Cytometry. 2001, 46: 79-84. 10.1002/cyto.1068.Imoto I, Tsuda H, Hirasawa A, Miura M, Sakamoto M, Hirohashi S, Inazawa J: Expression of cIAP1, a target for 11q22 amplification, correlates with resistance of cervical cancers to radiotherapy. Cancer Res. 2002, 62: 4860-4866.Dai Z, Zhu WG, Morrison CD, Brena RM, Smiraglia DJ, Raval A, Wu YZ, Rush LJ, Ross P, Molina JR: A comprehensive search for DNA amplification in lung cancer identifies inhibitors of apoptosis cIAP1 and cIAP2 as candidate oncogenes. Hum Mol Genet. 2003, 12: 791-801. 10.1093/hmg/ddg083.Bashyam MD, Bair R, Kim YH, Wang P, Hernandez-Boussard T, Karikari CA, Tibshirani R, Maitra A, Pollack JR: Array-based comparative genomic hybridization identifies localized DNA amplifications and homozygous deletions in pancreatic cancer. Neoplasia. 2005, 7: 556-562. 10.1593/neo.04586.Helias-Rodzewicz Z, Perot G, Chibon F, Ferreira C, Lagarde P, Terrier P, Coindre JM, Aurias A: YAP1 And VGLL3, encoding two cofactors of TEAD transcription factors, are amplified and overexpressed in a subset of soft tissue sarcomas. Genes Chromosomes Cancer. 2010, 49: 1161-1171. 10.1002/gcc.20825.Fernandez LA, Northcott PA, Dalton J, Fraga C, Ellison D, Angers S, Taylor MD, Kenney AM: YAP1 Is amplified and up-regulated in hedgehog-associated medulloblastomas and mediates sonic hedgehog-driven neural precursor proliferation. Genes Dev. 2009, 23: 2729-2741. 10.1101/gad.1824509.Muramatsu T, Imoto I, Matsui T, Kozaki K, Haruki S, Sudol M, Shimada Y, Tsuda H, Kawano T, Inazawa J: YAP is a candidate oncogene for esophageal squamous cell carcinoma. Carcinogenesis. 2010, 32: 389-398.Chigurupati S, Venkataraman R, Barrera D, Naganathan A, Madan M, Paul L, Pattisapu JV, Kyriazis GA, Sugaya K, Bushnev S: Receptor channel TRPC6 is a key mediator of notch-driven glioblastoma growth and invasiveness. Cancer Res. 2010, 70: 418-427. 10.1158/0008-5472.CAN-09-2654.Ding X, He Z, Zhou K, Cheng J, Yao H, Lu D, Cai R, Jin Y, Dong B, Xu Y, Wang Y: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma. J Natl Cancer Inst. 2010, 102: 1052-1068. 10.1093/jnci/djq217.Lansford CDGR, Bier H: Head and neck cancers. 1999, Dordrecht: Kluwer Academic Pressvan den Ijssel P, Tijssen M, Chin SF, Eijk P, Carvalho B, Hopmans E, Holstege H, Bangarusamy DK, Jonkers J, Meijer GA: Human and mouse oligonucleotide-based array CGH. Nucleic Acids Res. 2005, 33: e192-10.1093/nar/gni191.Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta C(T)) method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.Gollin SM: Chromosomal alterations in squamous cell carcinomas of the head and neck: window to the biology of disease. Head Neck. 2001, 23: 238-253. 10.1002/1097-0347(200103)23:33.0.CO;2-H.Smeets SJ, Braakhuis BJ, Abbas S, Snijders PJ, Ylstra B, van de Wiel MA, Meijer GA, Leemans CR, Brakenhoff RH: Genome-wide DNA copy number alterations in head and neck squamous cell carcinomas with or without oncogene-expressing human papillomavirus. Oncogene. 2006, 25: 2558-2564. 10.1038/sj.onc.1209275.Snijders AM, Schmidt BL, Fridlyand J, Dekker N, Pinkel D, Jordan RC, Albertson DG: Rare amplicons implicate frequent deregulation of cell fate specification pathways in oral squamous cell carcinoma. Oncogene. 2005, 24: 4232-4242. 10.1038/sj.onc.1208601.Canel M, Secades P, Garzon-Arango M, Allonca E, Suarez C, Serrels A, Frame M, Brunton V, Chiara MD: Involvement of focal adhesion kinase in cellular invasion of head and neck squamous cell carcinomas via regulation of MMP-2 expression. Br J Cancer. 2008, 98: 1274-1284. 10.1038/sj.bjc.6604286.Canel M, Secades P, Rodrigo JP, Cabanillas R, Herrero A, Suarez C, Chiara MD: Overexpression of focal adhesion kinase in head and neck squamous cell carcinoma is independent of fak gene copy number. Clin Cancer Res. 2006, 12: 3272-3279. 10.1158/1078-0432.CCR-05-1583.Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe JP, Tong F: A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell. 2006, 10: 515-527. 10.1016/j.ccr.2006.10.008.Jarvinen AK, Autio R, Kilpinen S, Saarela M, Leivo I, Grenman R, Makitie AA, Monni O: High-resolution copy number and gene expression microarray analyses of head and neck squamous cell carcinoma cell lines of tongue and larynx. Genes Chromosomes Cancer. 2008, 47: 500-509. 10.1002/gcc.20551.Lockwood WW, Chari R, Coe BP, Girard L, Macaulay C, Lam S, Gazdar AF, Minna JD, Lam WL: DNA amplification is a ubiquitous mechanism of oncogene activation in lung and other cancers. Oncogene. 2008, 27: 4615-4624. 10.1038/onc.2008.98.Weber A, Hengge UR, Stricker I, Tischoff I, Markwart A, Anhalt K, Dietz A, Wittekind C, Tannapfel A: Protein microarrays for the detection of biomarkers in head and neck squamous cell carcinomas. Hum Pathol. 2007, 38: 228-238. 10.1016/j.humpath.2006.07.012.Pacheco MM, Kowalski LP, Nishimoto IN, Brentani MM: Differential expression of c-jun and c-fos mRNAs in squamous cell carcinoma of the head and neck: associations with uPA, gelatinase B, and matrilysin mRNAs. Head Neck. 2002, 24: 24-32. 10.1002/hed.10009.Xie M, Sun Y, Li Y: Expression of matrix metalloproteinases in supraglottic carcinoma and its clinical implication for estimating lymph node metastases. Laryngoscope. 2004, 114: 2243-2248. 10.1097/01.mlg.0000149467.18822.59.Werner JA, Rathcke IO, Mandic R: The role of matrix metalloproteinases in squamous cell carcinomas of the head and neck. Clin Exp Metastasis. 2002, 19: 275-282. 10.1023/A:1015531319087.Zhang L, Ye DX, Pan HY, Wei KJ, Wang LZ, Wang XD, Shen GF, Zhang ZY: Yes-associated protein promotes cell proliferation by activating Fos related activator-1 in oral squamous cell carcinoma. Oral Oncol. 2011, 47: 693-697. 10.1016/j.oraloncology.2011.06.003.Yokoyama T, Osada H, Murakami H, Tatematsu Y, Taniguchi T, Kondo Y, Yatabe Y, Hasegawa Y, Shimokata K, Horio Y: YAP1 Is involved in mesothelioma development and negatively regulated by Merlin through phosphorylation. Carcinogenesis. 2008, 29: 2139-2146. 10.1093/carcin/bgn200.Diep CH, Zucker KM, Hostetter G, Watanabe A, Hu C, Munoz RM, Von Hoff DD, Han H: Down-regulation of Yes associated protein 1 expression reduces cell proliferation and clonogenicity of pancreatic cancer cells. PLoS One. 7: e32783-Kang W, Tong JH, Chan AW, Lee TL, Lung RW, Leung PP, So KK, Wu K, Fan D, Yu J: Yes-associated protein 1 exhibits oncogenic property in gastric cancer and its nuclear accumulation associates with poor prognosis. Clin Cancer Res. 2011, 17: 2130-2139. 10.1158/1078-0432.CCR-10-2467.Overholtzer M, Zhang J, Smolen GA, Muir B, Li W, Sgroi DC, Deng CX, Brugge JS, Haber DA: Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon. Proc Natl Acad Sci U S A. 2006, 103: 12405-12410. 10.1073/pnas.0605579103.Guilbert A, Dhennin-Duthille I, Hiani YE, Haren N, Khorsi H, Sevestre H, Ahidouch A, Ouadid-Ahidouch H: Expression of TRPC6 channels in human epithelial breast cancer cells. BMC Cancer. 2008, 8: 125-10.1186/1471-2407-8-125.Yue D, Wang Y, Xiao JY, Wang P, Ren CS: Expression of TRPC6 in benign and malignant human prostate tissues. Asian J Androl. 2009, 11: 541-547. 10.1038/aja.2009.53.Cai R, Ding X, Zhou K, Shi Y, Ge R, Ren G, Jin Y, Wang Y: Blockade of TRPC6 channels induced G2/M phase arrest and suppressed growth in human gastric cancer cells. Int J Cancer. 2009, 125: 2281-2287. 10.1002/ijc.24551.Shi Y, Ding X, He ZH, Zhou KC, Wang Q, Wang YZ: Critical role of TRPC6 channels in G2 phase transition and the development of human oesophageal cancer. Gut. 2009, 58: 1443-1450. 10.1136/gut.2009.181735.Thebault S, Flourakis M, Vanoverberghe K, Vandermoere F, Roudbaraki M, Lehen’kyi V, Slomianny C, Beck B, Mariot P, Bonnal JL: Differential role of transient receptor potential channels in Ca2+ entry and proliferation of prostate cancer epithelial cells. Cancer Res. 2006, 66: 2038-2047. 10.1158/0008-5472.CAN-05-0376.El Boustany C, Bidaux G, Enfissi A, Delcourt P, Prevarskaya N, Capiod T: Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation. Hepatology. 2008, 47: 2068-2077. 10.1002/hep.22263